CN117363658A - Clcn6基因点突变小鼠动物模型的构建方法及应用 - Google Patents
Clcn6基因点突变小鼠动物模型的构建方法及应用 Download PDFInfo
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Abstract
本发明提供了一种Clcn6基因变异相关早发性神经退行性疾病小鼠模型的构建方法与应用,属于生物医药技术领域。本发明基于CRISPR/Cas9基因编辑技术构建Clcn6基因点突变小鼠模型,构建方法为:设计并体外转录gRNA,同时构建Donor vector,将Cas9、gRNA、Donor vector同时注射到小鼠的受精卵中,获得携带病人等位点突变的Clcn6基因p.E200A敲入小鼠模型;并进行基因型鉴定。本发明首次成功建立了携带病人等位点突变(p.E200A)的Clcn6基因敲入(Clcn6E200A/+)小鼠模型,且未检测到脱靶效应。Clcn6E200A/+小鼠模型不仅再现了CLCN6p.E200A病人临床症状,而且复刻了神经元蜡样脂褐素沉积症的临床表现和组织病理特点,为CLCN6基因变异相关疾病机制研究及药物研发提供了可靠的动物模型。
Description
技术领域
本发明属于生物医药领域,涉及一种Clcn6基因点突变小鼠动物模型的构建方法及应用。
背景技术
电压门控氯离子通道和氯转运体(Voltage-gated Chloride Channel andTransporter,CLC)家族有9个成员,分为氯离子通道和氯转运体两大类。其中CLCN1、CLCN2、CLCNka、CLCNkb编码的CIC-1、CIC-2、CIC-Ka、CIC-Kb为细胞膜上的氯离子通道,在电兴奋性、胞内外离子稳态、跨上皮转运等方面发挥作用;CLCN3至CLCN7编码的ClC-3至ClC-7为内体/溶酶体膜上的2Cl-/H+反向转运体,主要影响囊泡离子组成、内吞作用及溶酶体功能。随着CLC家族模型动物的研究进展和病人中不断鉴定出CLC家族成员基因致病性变异,其与疾病的关系逐渐被发现。除了主要在肾脏表达的ClC-5,其他定位于内体/溶酶体上的2Cl-/H+反向转运体功能障碍均可导致严重神经系统疾病,比如CLCN4基因突变可导致人类出现智力障碍和癫痫表型,CLCN6基因突变可引起人类早发性神经退行性疾病,以及CLCN7基因突变引起人类和小鼠溶酶体贮积病。
ClC-6是晚期内体膜上的2Cl-/H+交换体,特异性的表达于神经系统,其将Cl-从细胞质逆浓度梯度转运到晚期内体,同时将H+由晚期内体转运至细胞质。内体、溶酶体的酸化主要由其膜上的囊泡型质子泵(Vacuolar H+-Adenosine Triphosphatase,v-ATPase)完成。ClC-6向晚期内体腔内转运带负电荷的Cl-可以中和v-ATPase在转运H+过程中建立的腔内阳性膜电势,从而易化内体、溶酶体的酸化。因此,ClC-6生理功能可能与晚期内体的酸化、Cl-的累积和细胞内物质运输有关。2006年,德国Thomas团队研究发现,纯合Clcn6-/-小鼠可正常存活,无肉眼及病理可见的异常表型。因此,CLCN6基因一直不被关注,在OMIM上也一直未与人类疾病关联。直到2021年,本团队以及Thomas团队报道了共4例CLCN6杂合变异的病例,CLCN6基因才与早发性神经退行性疾病关联。我们团队和Thomas团队报道的CLCN6杂合变异病人均存在严重的神经系统表型,这与纯合Clcn6-/-小鼠仅有轻度的行为学异常和杂合的Clcn6+/-小鼠正常表型形成鲜明对比,提示CLCN6基因变异可能不是通过功能丧失,而是通过功能获得的机制致病。但以上结果基于家系资料和模式细胞研究,未进行深入的功能研究及模式动物。为了明确CLCN6基因变异导致早发性神经退行性疾病致病机制,我们团队采用CRISPR/Cas9基因编辑技术成功构建全球首个Clcn6(CLCN6的同源基因)基因点突变小鼠(Clcn6E200A/+)模型。
近年来CRISPR/Cas9技术获得了快速发展。该技术是基于对细菌和古细菌中的免疫系统改造而建立,通过sgRNA介导核酸内切酶Cas9蛋白进行目标DNA序列识别并造成DNA双链断裂,促进以同源重组或非同源末端连接方式修复受损DNA,从而对目标位点实现基因的定点敲除、敲入以及基因修正等多种修饰。使用CRISPR/Cas9技术进行基因编辑需要两个关键因素,首先是有效的sgRNA引导序列,再就是Cas9蛋白的存在。与锌指核酸酶(ZFN)技术和类转录样效应因子核酸酶(TALEN)技术相比,因其靶向编辑目标基因的特异性、高效性和设计的简便性等诸多优点得到越来越广泛的应用,在细菌、哺乳动物细胞以及斑马鱼、小鼠、大鼠等都表现出很强的基因组编辑活性。
因此,建立特异性高,分子构建简单,流程短的CRISPR/Cas9基因编辑技术构建Clcn6基因点突变小鼠动物模型及其应用,具有十分重要的社会意义和经济价值。
发明内容
本发明旨在基于CRISPR/Cas9技术提供一种Clcn6基因点突变小鼠动物模型的构建方法及应用;本发明利用CRISPR/Cas9技术,通过同源重组的原理,对靶位点进行基因修饰。具体过程如下:设计并体外转录gRNA,同时构建同源重组载体(Donor vector);将Cas9,gRNA,Donor vector同时注射到小鼠的受精卵中;Cas9蛋白在gRNA引导下结合到靶位点进而造成DNA双链断裂,Donor vector通过同源重组修复断裂的双链,从而实现对靶位点的基因修饰。获得了全球首个携带病人变异的Clcn6(CLCN6的同源基因)基因点突变小鼠(Clcn6E200A/+)模型,为进一步研究Clcn6的功能奠定了良好的基础。
为了达到上述目的,本发明提供的技术方案为:
第一方面,本发明涉及一种Clcn6基因点突变小鼠动物模型的构建方法,所述构建方法包括如下步骤:
步骤一,设计并体外转录gRNA,同时构建同源重组载体Donor vector,通过胚胎显微注射移植法将Cas9、gRNA、Donor vector同时注射到C57BL/6J野生型小鼠的受精卵中,获得Clcn6基因点突变Clcn6E200A/+小鼠;
步骤二,对Clcn6基因点突变小鼠动物模型进行基因型鉴定。
优选的,步骤一包括以下步骤:
S11,设计并体外转录gRNA,同时构建同源重组载体Donor vector;
S12,通过胚胎显微注射移植法将Cas9、gRNA、Donor vector同时注射到C57BL/6J野生型小鼠的受精卵中;
S13,经胚胎移植,获得F0小鼠,通过PCR,测序确认,共获得如下同源重组的阳性F0代小鼠;
S14,将得到的阳性F0代雄性小鼠分别与野生型雌性小鼠进行交配,获得阳性F1代小鼠,或者将F0代雄性小鼠的精子与C57BL/6J野生型小鼠卵母细胞进行体外受精并移植入假孕母鼠体内,获得阳性F1代小鼠,通过PCR和DNA测序筛选出Clcn6基因点突变小鼠动物模型。
优选的,步骤二包括以下步骤:
S21,提取F0代小鼠尾部DNA,经PCR和DNA测序确认;
S22,提取F1代小鼠尾部DNA,根据鉴定策略示意图,经PCR和DNA测序确认。
所述鉴定策略示意图如图2所示。
其中,野生型:测序结果仅存在Wildtype序列;杂合子:测序结果同时存在Wildtype&Mutation序列;纯合子:测序结果仅存在Mutation序列。
优选的,所述PCR扩增采用Clcn6-seq-F1与Clcn6-seq-R1引物对;引物Clcn6-seq-F1的序列如SEQ ID NO.1所示;引物Clcn6-seq-R1的序列如SEQ ID NO.2所示。
所述SEQ ID NO.1的引物序列为TCGGCAGTCCTACCTCTAGTTC。
所述SEQ ID NO.2的引物序列为AGACTCAGCAGTGATGCTATGC。
第二方面,本发明涉及一种基于CRISPR/Cas9基因编辑技术构建Clcn6基因点突变小鼠模型,设计特异性的sgRNA,所述sgRNA序列如SEQ ID NO:3所示。
所述SEQ ID NO.3的sgRNA序列为GAAGGCCCCATGATCCACAG。
第三方面,本发明涉及一种Clcn6基因靶向载体,所述载体是基于
CRISPR/Cas9系统的sgRNA表达载体,sgRNA的作用位点的序列如SEQ ID NO:3所示。
第四方面,本发明涉及一种前述的CRISPR/Cas9基因编辑技术,针对Clcn6基因设计和构建gRNA与Cas表达质粒,在构建Clcn6基因变异相关早发性神经退行性疾病或其他神经系统疾病相关的小鼠动物模型中的用途。
上述的Clcn6基因点突变小鼠动物模型可用于早发性神经退行性疾病或其他神经系统疾病相关的研究。
本发明具有如下有益效果:
本发明基于成簇规律间隔短回文重复序列(Clustered Regularly InterspacedShort Palindromic Repeats,CRISPR/Cas9)基因-编辑技术构建Clcn6基因点突变小鼠模型,首次成功建立了携带病人等位点突变(p.E200A)的Clcn6基因敲入(Clcn6E220A/+)小鼠动物模型,且未检测到脱靶效应。我们构建的Clcn6E220A/+小鼠模型表现出后肢抱茎、全身震颤、共济失调、癫痫发作、运动障碍、过早死亡等严重的神经系统表型,不仅再现了CLCN6p.E200A病人临床症状,而且复刻了神经元蜡样脂褐素沉积症的临床表现和组织病理特点。因此,本发明构建的Clcn6E200A/+小鼠模型为深入探索与揭示Clcn6基因变异相关神经退行性疾病的病理学机制,以及开展潜在药物的临床前研发等基础和转化研究提供了便捷、可靠的动物模型。为早发性神经退行性疾病或其他神经系统疾病的相关研究提供新的研究方向。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1:Clcn6基因点突变小鼠策略示意图;
图2:F1代小鼠鉴定策略示意图;(其中,野生型:测序结果仅存在Wildtype序列;杂合子:测序结果同时存在Wildtype&Mutation序列;纯合子:测序结果仅存在Mutation序列)
图3:F1代小鼠PCR检测结果;(注:数字为鼠尾号,WT为C57BL/6J野生型,N为negative空白对照,M为DNA Marker)
图4:F1代小鼠基因部分测序结果;
图5:Clcn6基因点突变小鼠动物模型效果图1;
图6:Clcn6基因点突变小鼠动物模型效果图2。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。应当指出的是,对本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干调整和改进。这些都属于本发明的保护范围。
本发明中,Donor oligo:金斯瑞合成,crRNA:金斯瑞合成,tracRNA:IDT购买;
一、Clcn6基因点突变小鼠的构建方案,示意图如图1所示;
(1)构建针对Clcn6基因的特异性靶点sgRNA。线性化及纯化DNA并在体外转录为gRNA,同时构建同源重组载体(Donor vector)。所述Clcn6的sgRNA序列如SEQ ID NO.3所示;gRNA序列信息如下所示:
表1-1gRNA序列信息
(2)将步骤(1)所述的gRNA、Cas9、Donor vector,同时显微注射到受精卵中,取注射后存活的受精卵移植到代孕母鼠子宫,孕育出生的小鼠即为F0代小鼠;
所述Donor vector序列信息如下所示:
GTTGCTGAAAGGTTTACATAGCAAAATCTAGGCCAGGGGCCCTCTGCTGACTGTGCCCCTCTTTTCTCCCCACTCCCAGGGCTCTTTGTGGGGAAAGCAGGCCCCATGATCCATTCCGGTGCTGTGGTAGGAGCTGGCCTCCCTCAGGTAAGGATTGGTGGGCCAGGGGATGCGTTCCTTAGAATGGGTCTCTTGCATAGGCCCAGCAGATGTGATATCCCTCATGC
其中,下划线碱基GCA为目标点突变序列,下划线碱基TTCC为突变PAM而做的同义突变碱基;
(3)待F0代小鼠出生10天后剪尾提取DNA进行PCR鉴定,并进行DNA测序。得到的同源重组的阳性F0代小鼠,具体信息如下:
ID | Gender | color | Gty | DOB | Gen |
169 | ♂ | B | 阳性 | 2018/4/14 | F0 |
(4)把上述步骤(3)得到的F0代阳性小鼠分别与4只野生型雌性小鼠进行交配,合笼约1个月后,4只野生型雌性小鼠一直未怀孕,故在F0代雄性小鼠3月龄时处死,并快速取精子,然后将F0代雄性小鼠的精子与C57BL/6J野生型小鼠卵母细胞进行体外受精并移植入假孕母鼠体内,获得阳性F1代小鼠,即为Clcn6基因点突变小鼠动物模型。
二、F1代小鼠PCR鉴定方法及结果:
(1)鉴定策略示意图:如图2。
(2)引物信息如表2:
表2
(3)测序方案如下表3:
表3
(4)PCR条件如下所示:
(5)阳性F1代小鼠信息如下:
ID | Gender | color | Gty | DOB | Gen | F |
1 | ♂ | B | KI/wt | 2018/7/29 | F1 | 169# |
2 | ♂ | B | KI/wt | 2018/7/29 | F1 | 169# |
4 | ♂ | B | KI/wt | 2018/7/29 | F1 | 169# |
6 | ♀ | B | KI/wt | 2018/7/29 | F1 | 169# |
8 | ♂ | B | KI/wt | 2018/7/29 | F1 | 169# |
10 | ♀ | B | KI/wt | 2018/7/29 | F1 | 169# |
注:两对引物需要分别进行PCR。
(6)F1代小鼠鉴定结果:根据PCR策略示意图,经PCR和测序确认,1,2,4,6,8,10号为阳性F1代小鼠。电泳图如图3所示。
其中,测序结果如图4所示。
三、F1代小鼠(Clcn6E200A/+小鼠)动物模型效果
将本发明的Clcn6E200A/+小鼠进行动物模型效果实验,实验结果如图5、图6所示。
由图5可知,Clcn6E200A/+小鼠表型(A)生存分析显示,Clcn6E200A/+在3-7月龄死亡,而Clcn6-/-小鼠可正常存活。(B)与Clcn6+/+小鼠相比,Clcn6E200A/+24周前体重无明显差异,24周时体重下降,而Clcn6-/-小鼠始终无明显差异。(C)尾巴悬吊时Clcn6+/+和Clcn6-/-小鼠后肢外展,而Clcn6E200A/+小鼠后肢抱茎。(D)头颅MRI显示,Clcn6E200A/+和Clcn6-/-小鼠均存在双侧侧脑室的扩张,但仅小鼠Clcn6E200A/+有脑容量减小。(E)视频脑电图显示,Clcn6E200A/+小鼠存在大量的尖波和尖波节律发放,Clcn6-/-小鼠仅有少量的低波幅的尖波发放。(F)视网膜电流图分析显示,Clcn6E200A/+和Clcn6-/-小鼠均有视网膜功能损害,Clcn6E200A/+小鼠的视网膜功能损害更严重。
由图6可知:Clcn6E200A/+小鼠组织病理特点(A)大脑形态学显示,Clcn6E200A/+小鼠存在全脑萎缩,而Clcn6-/-小鼠脑体积无明显改变。(B)海马尼氏染色显示,Clcn6E200A/+小鼠存在进行性海马神经元丢失,而Clcn6-/-小鼠无海马神经元丢失。(C)小脑苏木精伊红染色显示,Clcn6E200A/+小鼠存在进行性海马神经元丢失,而Clcn6-/-小鼠无海马神经元丢失。(D)Luxol Fast Blue染色显示,Clcn6E200A/+小鼠大脑皮层存在严重的髓鞘缺陷,Clcn6-/-小鼠轻度髓鞘损害。(E-F)免疫荧光分析显示,Clcn6E200A/+小鼠生后35天(E)和5月龄(F)脑组织均存在胶质纤维酸性蛋白(GFAP)标记的星形胶质细胞和离子钙结合衔接分子1(IBA-1)标记的小胶质细胞的增生,而Clcn6-/-小鼠无胶质细胞增生。
故,本发明的Clcn6E200A/+小鼠模型表现出严重神经系统缺陷表现,包括后肢抱茎、全身震颤、共济失调、癫痫发作、运动障碍、过早死亡、进行性运动障碍、视觉损害、过早死亡、全脑萎缩、溶酶体内自荧光脂褐素物质贮积、胶质细胞增生和进行性神经元丢失等,不仅再现了CLCN6 p.E200A病人临床症状,而且复刻了神经元蜡样脂褐素沉积症的临床表现和组织病理特点。
本发明基于成簇规律间隔短回文重复序列(Clustered Regularly InterspacedShort Palindromic Repeats,CRISPR/Cas9)基因-编辑技术构建Clcn6基因点突变小鼠模型,首次成功建立了携带病人等位点突变(p.E200A)的Clcn6基因敲入(Clcn6E220A/+)小鼠动物模型,且未检测到脱靶效应。我们构建的Clcn6E220A/+小鼠模型表现出后肢抱茎、全身震颤、共济失调、癫痫发作、运动障碍、过早死亡等严重的神经系统表型,不仅再现了CLCN6p.E200A病人临床症状,而且复刻了神经元蜡样脂褐素沉积症的临床表现和组织病理特点。因此,本发明构建的Clcn6E200A/+小鼠模型为深入探索与揭示Clcn6基因变异相关神经退行性疾病的病理学机制,以及开展潜在药物的临床前研发等基础和转化研究提供了便捷、可靠的动物模型。
本发明的方法已经通过较佳实施例进行了描述,相关人员明显能在本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。本领域技术人员可以借鉴本文内容,适当改进参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明内。
Claims (7)
1.一种Clcn6基因敲入点突变小鼠动物模型的构建方法,其特征在于,所述构建方法包括如下步骤:
步骤一,设计并体外转录gRNA,同时构建同源重组载体Donor vector,通过胚胎显微注射移植法将Cas9、gRNA、Donor vector同时注射到C57BL/6J野生型小鼠的受精卵中,获得Clcn6基因点突变小鼠模型;
步骤二,对Clcn6基因点突变小鼠动物模型进行基因型鉴定。
2.根据权利要求1所述的Clcn6基因点突变小鼠动物模型的构建方法,其特征在于,步骤一包括以下步骤:
S1,设计并体外转录gRNA,同时构建同源重组载体Donor vector;
S2,通过胚胎显微注射移植法将Cas9、gRNA、Donor vector同时注射到C57BL/6J野生型小鼠的受精卵中;
S3,经胚胎移植,获得F0小鼠,通过PCR和DNA测序确认基因型,获得雄性同源重组的阳性F0代小鼠;
S4,将得到的阳性F0代雄性小鼠分别与野生型雌性小鼠进行交配,获得阳性F1代小鼠,或者将F0代雄性小鼠的精子与C57BL/6J野生型小鼠卵母细胞进行体外受精并移植入假孕母鼠体内,获得阳性F1代小鼠,通过PCR和DNA测序筛选出Clcn6基因点突变小鼠动物模型。
3.根据权利要求1或2所述的Clcn6基因点突变小鼠动物模型的构建方法,其特征在于,步骤二包括以下步骤:
S5,提取F0代小鼠尾部DNA,经PCR和DNA测序确认;
S6,提取F1代小鼠尾部DNA,根据鉴定策略示意图,经PCR和DNA测序确认基因型。
4.根据权利要求3所述的Clcn6基因点突变小鼠动物模型的构建方法,其特征在于,所述PCR扩增采用Clcn6-seq-F1与Clcn6-seq-R1引物对;引物Clcn6-seq-F1的序列如SEQ IDNO.1所示;引物Clcn6-seq-R1的序列如SEQ ID NO.2所示。
5.根据权利要求3所述的Clcn6基因点突变小鼠动物模型的构建方法,其特征在于,所述F1代小鼠基因组部分序列如图4所示。
6.一种Clcn6基因靶向载体,其特征在于,所述载体是基于CRISPR/Cas9系统的sgRNA表达载体,sgRNA的作用位点的序列如SEQ ID NO:3所示。
7.一种如权利要求6所述的sgRNA在构建早发性神经退行性疾病或其他神经系统综合征相关的基因点突变小鼠动物模型中的用途。
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